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Endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe as well as synthesis method and application thereof

A nitroreductase, fluorescent probe technology, applied in fluorescence/phosphorescence, chemical instruments and methods, luminescent materials, etc., to achieve the effects of high yield, high sensitivity and simple synthesis method

Inactive Publication Date: 2017-12-08
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the current lack of endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probes, the present invention provides an endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe

Method used

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  • Endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe as well as synthesis method and application thereof
  • Endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe as well as synthesis method and application thereof
  • Endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe as well as synthesis method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Synthesis of fluorescent probes.

[0045] In a 25 mL round-bottomed flask, slowly add ethylenediamine (compound 1) 1 mmol dropwise to toluene (5 mL) at 40 °C, add 1.0 mmol p-toluenesulfonyl chloride (compound 2) while fully stirring, and heat After refluxing for 3-4 h, cool to room temperature, add 5ml of 2M HCl aqueous solution to the reaction, remove the filter residue by suction filtration, neutralize the filtrate with NaOH until a large amount of white solids are precipitated, filter under reduced pressure, and dry the solid in vacuum to obtain a solid N - (2 - aminoethyl) - 4 - methyl sulfonamide (compound 3), yield: 72%. The product was directly subjected to the next reaction without purification.

[0046] In a 100 mL round bottom flask, add 243 mg 4-nitronaphthalene dicarboxylate (compound 4) (1 mmol) and 20 mL ethanol, add 0.1 mL triethylamine and 321 mg N-(2-aminoethyl )-4-methylbenzenesulfonamide (compound 3) (1.5 mmol), then heated to reflux for 6...

Embodiment 2

[0048] Example 2 Fluorescence Probe Na-ER-NTR Absorption Spectrum Test

[0049] The fluorescent probe Na-ER-NTR in Example 1 was weighed and prepared into a 1 mM stock solution with dimethyl sulfoxide (DMSO).

[0050] Take 25 μL of the probe mother solution and add it to three identical 5 mL volumetric flasks, add 225 μL of DMSO solution, and add reduced coenzyme I (NADH, final concentration 300 μM) to two of them. One of the two coenzyme-added volumetric flasks was added with phosphate buffered solution of nitroreductase (final concentration 2.0 μg / mL), and the other was not. All the three volumetric flasks were made to volume with phosphate buffer solution (0.01M PBS, pH = 7.4) and reacted at 37°C for 60 min, and then the absorption spectrum test was carried out, with the wavelength as the abscissa and the absorbance as the ordinate. figure 2 . Depend on figure 2 It can be seen that the absorption intensity of a single probe is very small, and there is a weak absorption...

Embodiment 3

[0051] Example 3 Kinetic test of the interaction between fluorescent probe Na-ER-NTR and nitroreductase.

[0052] Dilute the fluorescent probe master solution in Example 2 into a 5 μM probe working solution for later use.

[0053] Prepare a series of solutions of nitroreductase at concentrations of 0, 0.5, 1.0, 1.5, 2.5, and 5.0 μg / mL in PBS with a concentration of 0.01 M at pH = 7.4.

[0054] The probe working solution was mixed with different concentrations of nitroreductase, and the final concentration of NADH was 300 μM, and then the fluorescence detection (λ ex = 440 nm, λ em = 545 nm), tested every 3 min for 80 min, recorded the fluorescence intensity in each system over time, and established a curve of fluorescence intensity over time, as Figure 4 As shown, the fluorescence intensity of the reaction system gradually increased, and tended to be stable at nearly 60 min, and the fluorescence intensity of the reaction system reached saturation.

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Abstract

The invention provides an endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe, wherein the chemical name is 4-nitro-N-(2-(4-methyl benzenesulfonamido)ethyl)-naphthalimide. The endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe is synthesized by the following steps: enabling ethylenediamine to completely react with paratoluensulfonyl chloride to obtain N-(2-aminoethyl)-4-toluenesulfonamide; enabling 4-nitro-1,8-naphthalic anhydride to react with the N-(2-aminoethyl)-4-toluenesulfonamide to obtain the endoplasmic reticulum-targeted two-photon nitroreductase fluorescent probe. The invention also provides application of the fluorescent probe in detecting nitroreductase activity in a solution and a biological body by use of single-proton or two-photon fluorescence. The probe synthesis method provided by the invention is simple and realizes high yield; the probe can be positioned on the endoplasmic reticulum to perform two-photon fluorescence detection on cells or tissues, and also has high sensitivity and can resist the interference of multiple interfering substances.

Description

technical field [0001] The invention relates to a fast-response nitroreductase fluorescent probe, in particular to an endoplasmic reticulum-targeted two-photon effect fluorescent probe and its synthesis method and application, belonging to the field of organic small molecule fluorescent probes. Background technique [0002] Hypoxia is an important feature of the occurrence and development of malignant tumors, mainly due to the unlimited growth and metabolism of tumor cells, which increases oxygen consumption, resulting in insufficient blood oxygen supply in the tumor area. Medical research shows that the tumor hypoxic area generally refers to the cell population in the 10-20 μm area of ​​the tumor blood vessel, and the cells in this area are also called hypoxic cells. Because hypoxic cells do not have sufficient oxygen supply, the cells are in a relatively quiescent state where they cannot proliferate or die, and this state is generally at the boundary between G1 and S phase...

Claims

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Application Information

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IPC IPC(8): C09K11/06C07D221/14G01N21/64
CPCC09K11/06C07D221/14C09K2211/1007C09K2211/1029G01N21/6486
Inventor 林伟英唐永和徐安马燕燕
Owner UNIV OF JINAN
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